Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.630
Filtrar
1.
Nat Commun ; 12(1): 847, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558503

RESUMO

A large G4C2-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Neuronal degeneration associated with this expansion arises from a loss of C9orf72 protein, the accumulation of RNA foci, the expression of dipeptide repeat (DPR) proteins, or all these factors. We report the discovery of a new targeting sequence that is common to all C9orf72 transcripts but enables preferential knockdown of repeat-containing transcripts in multiple cellular models and C9BAC transgenic mice. We optimize stereopure oligonucleotides that act through this site, and we demonstrate that their preferential activity depends on both backbone stereochemistry and asymmetric wing design. In mice, stereopure oligonucleotides produce durable depletion of pathogenic signatures without disrupting protein expression. These oligonucleotides selectively protect motor neurons harboring C9orf72-expansion mutation from glutamate-induced toxicity. We hypothesize that targeting C9orf72 with stereopure oligonucleotides may be a viable therapeutic approach for the treatment of C9orf72-associated neurodegenerative disorders.


Assuntos
Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Mutação/genética , Oligonucleotídeos/química , Oligonucleotídeos/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Animais , Proteína C9orf72/química , Éxons/genética , Glutamatos/toxicidade , Íntrons/genética , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Processamento de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estereoisomerismo
2.
PLoS Biol ; 18(12): e3001002, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33362237

RESUMO

Nucleocytoplasmic transport (NCT) defects have been implicated in neurodegenerative diseases such as C9ORF72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). Here, we identify a neuroprotective pathway of like-Sm protein 12 (LSM12) and exchange protein directly activated by cyclic AMP 1 (EPAC1) that sustains the nucleocytoplasmic RAN gradient and thereby suppresses NCT dysfunction by the C9ORF72-derived poly(glycine-arginine) protein. LSM12 depletion in human neuroblastoma cells aggravated poly(GR)-induced impairment of NCT and nuclear integrity while promoting the nuclear accumulation of poly(GR) granules. In fact, LSM12 posttranscriptionally up-regulated EPAC1 expression, whereas EPAC1 overexpression rescued the RAN gradient and NCT defects in LSM12-deleted cells. C9-ALS patient-derived neurons differentiated from induced pluripotent stem cells (C9-ALS iPSNs) displayed low expression of LSM12 and EPAC1. Lentiviral overexpression of LSM12 or EPAC1 indeed restored the RAN gradient, mitigated the pathogenic mislocalization of TDP-43, and suppressed caspase-3 activation for apoptosis in C9-ALS iPSNs. EPAC1 depletion biochemically dissociated RAN-importin ß1 from the cytoplasmic nuclear pore complex, thereby dissipating the nucleocytoplasmic RAN gradient essential for NCT. These findings define the LSM12-EPAC1 pathway as an important suppressor of the NCT-related pathologies in C9-ALS/FTD.


Assuntos
Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Núcleo Celular/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética
3.
Proc Natl Acad Sci U S A ; 117(40): 25104-25115, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32958650

RESUMO

Maintaining the fidelity of nascent peptide chain (NP) synthesis is essential for proteome integrity and cellular health. Ribosome-associated quality control (RQC) serves to resolve stalled translation, during which untemplated Ala/Thr residues are added C terminally to stalled peptide, as shown during C-terminal Ala and Thr addition (CAT-tailing) in yeast. The mechanism and biological effects of CAT-tailing-like activity in metazoans remain unclear. Here we show that CAT-tailing-like modification of poly(GR), a dipeptide repeat derived from amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD)-associated GGGGCC (G4C2) repeat expansion in C9ORF72, contributes to disease. We find that poly(GR) can act as a mitochondria-targeting signal, causing some poly(GR) to be cotranslationally imported into mitochondria. However, poly(GR) translation on mitochondrial surface is frequently stalled, triggering RQC and CAT-tailing-like C-terminal extension (CTE). CTE promotes poly(GR) stabilization, aggregation, and toxicity. Our genetic studies in Drosophila uncovered an important role of the mitochondrial protease YME1L in clearing poly(GR), revealing mitochondria as major sites of poly(GR) metabolism. Moreover, the mitochondria-associated noncanonical Notch signaling pathway impinges on the RQC machinery to restrain poly(GR) accumulation, at least in part through the AKT/VCP axis. The conserved actions of YME1L and noncanonical Notch signaling in animal models and patient cells support their fundamental involvement in ALS/FTD.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/genética , Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Proteínas de Drosophila/genética , Demência Frontotemporal/genética , Metaloendopeptidases/genética , Proteínas Mitocondriais/genética , Proteoma/genética , Receptores Notch/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Arginina/genética , Expansão das Repetições de DNA/genética , Modelos Animais de Doenças , Drosophila melanogaster/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Células HEK293 , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Biossíntese de Proteínas , Ribossomos/genética , Ribossomos/metabolismo , Transdução de Sinais/genética
4.
Am J Hum Genet ; 107(3): 445-460, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32750315

RESUMO

Tandem repeats are proposed to contribute to human-specific traits, and more than 40 tandem repeat expansions are known to cause neurological disease. Here, we characterize a human-specific 69 bp variable number tandem repeat (VNTR) in the last intron of WDR7, which exhibits striking variability in both copy number and nucleotide composition, as revealed by long-read sequencing. In addition, greater repeat copy number is significantly enriched in three independent cohorts of individuals with sporadic amyotrophic lateral sclerosis (ALS). Each unit of the repeat forms a stem-loop structure with the potential to produce microRNAs, and the repeat RNA can aggregate when expressed in cells. We leveraged its remarkable sequence variability to align the repeat in 288 samples and uncover its mechanism of expansion. We found that the repeat expands in the 3'-5' direction, in groups of repeat units divisible by two. The expansion patterns we observed were consistent with duplication events, and a replication error called template switching. We also observed that the VNTR is expanded in both Denisovan and Neanderthal genomes but is fixed at one copy or fewer in non-human primates. Evaluating the repeat in 1000 Genomes Project samples reveals that some repeat segments are solely present or absent in certain geographic populations. The large size of the repeat unit in this VNTR, along with our multiplexed sequencing strategy, provides an unprecedented opportunity to study mechanisms of repeat expansion, and a framework for evaluating the roles of VNTRs in human evolution and disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Amiotrófica Lateral/genética , Evolução Molecular , Sequências de Repetição em Tandem/genética , Idoso , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Esclerose Amiotrófica Lateral/patologia , Expansão das Repetições de DNA/genética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Masculino , Repetições Minissatélites/genética , Fenótipo , Especificidade da Espécie
5.
Nat Commun ; 11(1): 3848, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737286

RESUMO

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by the degeneration of upper and lower motor neurons (MNs). We find a significant reduction of the retromer complex subunit VPS35 in iPSCs-derived MNs from ALS patients, in MNs from ALS post mortem explants and in MNs from SOD1G93A mice. Being the retromer involved in trafficking of hydrolases, a pathological hallmark in ALS, we design, synthesize and characterize an array of retromer stabilizers based on bis-guanylhydrazones connected by a 1,3-phenyl ring linker. We select compound 2a as a potent and bioavailable interactor of VPS35-VPS29. Indeed, while increasing retromer stability in ALS mice, compound 2a attenuates locomotion impairment and increases MNs survival. Moreover, compound 2a increases VPS35 in iPSCs-derived MNs and shows brain bioavailability. Our results clearly suggest the retromer as a valuable druggable target in ALS.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Hidrazonas/farmacologia , Neurônios Motores/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Proteínas de Transporte Vesicular/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Hidrazonas/síntese química , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/síntese química , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Relação Estrutura-Atividade , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Proteínas de Transporte Vesicular/metabolismo
6.
EBioMedicine ; 59: 102980, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32862101

RESUMO

BACKGROUND: Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease as well as Lou Gehrig's disease, is a progressive neurological disorder selectively affecting motor neurons with no currently known cure. Around 20% of the familial ALS cases arise from dominant mutations in the sod1 gene encoding superoxide dismutase1 (SOD1) enzyme. Aggregation of mutant SOD1 in familial cases and of wild-type SOD1 in at least some sporadic ALS cases is one of the known causes of the disease. Riluzole, approved in 1995 and edaravone in 2017 remain the only drugs with limited therapeutic benefits. METHODS: We have utilised the ebselen template to develop novel compounds that redeem stability of mutant SOD1 dimer and prevent aggregation. Binding modes of compounds have been visualised by crystallography. In vitro neuroprotection and toxicity of lead compounds have been performed in mouse neuronal cells and disease onset delay of ebselen has been demonstrated in transgenic ALS mice model. FINDING: We have developed a number of ebselen-based compounds with improvements in A4V SOD1 stabilisation and in vitro therapeutic effects with significantly better potency than edaravone. Structure-activity relationship of hits has been guided by high resolution structures of ligand-bound A4V SOD1. We also show clear disease onset delay of ebselen in transgenic ALS mice model holding encouraging promise for potential therapeutic compounds. INTERPRETATION: Our finding established the new generation of organo-selenium compounds with better in vitro neuroprotective activity than edaravone. The potential of this class of compounds may offer an alternative therapeutic agent for ALS treatment. The ability of these compounds to target cysteine 111 in SOD may have wider therapeutic applications targeting cysteines of enzymes involved in pathogenic and viral diseases including main protease of SARS-Cov-2 (COVID-19). FUNDING: Project funding was supported by the ALS Association grant (WA1128) and Fostering Joint International Research (19KK0214) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Compostos Organosselênicos/uso terapêutico , Superóxido Dismutase-1/metabolismo , Esclerose Amiotrófica Lateral/mortalidade , Esclerose Amiotrófica Lateral/patologia , Animais , Azóis/química , Azóis/metabolismo , Azóis/uso terapêutico , Betacoronavirus/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Dimerização , Modelos Animais de Doenças , Estabilidade Enzimática , Camundongos , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Compostos Organosselênicos/química , Compostos Organosselênicos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Superóxido Dismutase-1/genética , Taxa de Sobrevida , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo
7.
PLoS One ; 15(8): e0236804, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790801

RESUMO

OBJECTIVE: The ALS Functional Rating Scale-Revised (ALSFRS-R) is the most commonly utilized instrument to index bulbar function in both clinical and research settings. We therefore aimed to evaluate the diagnostic utility of the ALSFRS-R bulbar subscale and swallowing item to detect radiographically confirmed impairments in swallowing safety (penetration or aspiration) and global pharyngeal swallowing function in individuals with ALS. METHODS: Two-hundred and one individuals with ALS completed the ALSFRS-R and the gold standard videofluoroscopic swallowing exam (VFSE). Validated outcomes including the Penetration-Aspiration Scale (PAS) and Dynamic Imaging Grade of Swallowing Toxicity (DIGEST) were assessed in duplicate by independent and blinded raters. Receiver operator characteristic curve analyses were performed to assess accuracy of the ALSFRS-R bulbar subscale and swallowing item to detect radiographically confirmed unsafe swallowing (PAS > 3) and global pharyngeal dysphagia (DIGEST >1). RESULTS: Although below acceptable screening tool criterion, a score of ≤ 3 on the ALSFRS-R swallowing item optimized classification accuracy to detect global pharyngeal dysphagia (sensitivity: 68%, specificity: 64%, AUC: 0.68) and penetration/aspiration (sensitivity: 79%, specificity: 60%, AUC: 0.72). Depending on score selection, sensitivity and specificity of the ALSFRS-R bulbar subscale ranged between 34-94%. A score of < 9 optimized classification accuracy to detect global pharyngeal dysphagia (sensitivity: 68%, specificity: 68%, AUC: 0.76) and unsafe swallowing (sensitivity:78%, specificity:62%, AUC: 0.73). CONCLUSIONS: The ALSFRS-R bulbar subscale or swallowing item did not demonstrate adequate diagnostic accuracy to detect radiographically confirmed swallowing impairment. These results suggest the need for alternate screens for dysphagia in ALS.


Assuntos
Esclerose Amiotrófica Lateral/diagnóstico , Transtornos de Deglutição/diagnóstico , Idoso , Esclerose Amiotrófica Lateral/complicações , Esclerose Amiotrófica Lateral/patologia , Área Sob a Curva , Deglutição/fisiologia , Transtornos de Deglutição/etiologia , Feminino , Fluoroscopia , Humanos , Masculino , Pessoa de Meia-Idade , Curva ROC , Sensibilidade e Especificidade , Índice de Gravidade de Doença
8.
Nat Commun ; 11(1): 3753, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719333

RESUMO

Reactive astrocytes have been implicated in the pathogenesis of neurodegenerative diseases, including a non-cell autonomous effect on motor neuron survival in ALS. We previously defined a mechanism by which microglia release three factors, IL-1α, TNFα, and C1q, to induce neurotoxic astrocytes. Here we report that knocking out these three factors markedly extends survival in the SOD1G93A ALS mouse model, providing evidence for gliosis as a potential ALS therapeutic target.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Astrócitos/metabolismo , Complemento C1q/metabolismo , Progressão da Doença , Interleucina-1alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Complemento C3/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia , Superóxido Dismutase-1/metabolismo
9.
Neurology ; 95(8): e943-e952, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32646955

RESUMO

OBJECTIVE: To evaluate progressive white matter (WM) degeneration in amyotrophic lateral sclerosis (ALS). METHODS: Sixty-six patients with ALS and 43 healthy controls were enrolled in a prospective, longitudinal, multicenter study in the Canadian ALS Neuroimaging Consortium (CALSNIC). Participants underwent a harmonized neuroimaging protocol across 4 centers that included diffusion tensor imaging (DTI) for assessment of WM integrity. Three visits were accompanied by clinical assessments of disability (ALS Functional Rating Scale-Revised [ALSFRS-R]) and upper motor neuron (UMN) function. Voxel-wise whole-brain and quantitative tract-wise DTI assessments were done at baseline and longitudinally. Correction for site variance incorporated data from healthy controls and from healthy volunteers who underwent the DTI protocol at each center. RESULTS: Patients with ALS had a mean progressive decline in fractional anisotropy (FA) of the corticospinal tract (CST) and frontal lobes. Tract-wise analysis revealed reduced FA in the CST, corticopontine/corticorubral tract, and corticostriatal tract. CST FA correlated with UMN function, and frontal lobe FA correlated with the ALSFRS-R score. A progressive decline in CST FA correlated with a decline in the ALSFRS-R score and worsening UMN signs. Patients with fast vs slow progression had a greater reduction in FA of the CST and upper frontal lobe. CONCLUSIONS: Progressive WM degeneration in ALS is most prominent in the CST and frontal lobes and, to a lesser degree, in the corticopontine/corticorubral tracts and corticostriatal pathways. With the use of a harmonized imaging protocol and incorporation of analytic methods to address site-related variances, this study is an important milestone toward developing DTI biomarkers for cerebral degeneration in ALS. CLINICALTRIALSGOV IDENTIFIER: NCT02405182.


Assuntos
Esclerose Amiotrófica Lateral/diagnóstico por imagem , Córtex Cerebral/diagnóstico por imagem , Tratos Piramidais/diagnóstico por imagem , Substância Branca/diagnóstico por imagem , Adulto , Idoso , Esclerose Amiotrófica Lateral/patologia , Córtex Cerebral/patologia , Imagem de Tensor de Difusão , Progressão da Doença , Feminino , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Degeneração Neural/diagnóstico por imagem , Degeneração Neural/patologia , Neuroimagem/métodos , Estudos Prospectivos , Tratos Piramidais/patologia , Substância Branca/patologia
10.
Nat Commun ; 11(1): 3354, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620797

RESUMO

Expansion of an intronic (GGGGCC)n repeat region within the C9orf72 gene is a main cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). A hallmark of c9ALS/FTD is the accumulation of misprocessed RNAs, which are often targets of cellular RNA surveillance. Here, we show that RNA decay mechanisms involving upstream frameshift 1 (UPF1), including nonsense-mediated decay (NMD), are inhibited in c9ALS/FTD brains and in cultured cells expressing either of two arginine-rich dipeptide repeats (R-DPRs), poly(GR) and poly(PR). Mechanistically, although R-DPRs cause the recruitment of UPF1 to stress granules, stress granule formation is independent of NMD inhibition. Instead, NMD inhibition is primarily a result from global translational repression caused by R-DPRs. Overexpression of UPF1, but none of its NMD-deficient mutants, enhanced the survival of neurons treated by R-DPRs, suggesting that R-DPRs cause neurotoxicity in part by inhibiting cellular RNA surveillance.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteína C9orf72/genética , Demência Frontotemporal/genética , Degradação do RNAm Mediada por Códon sem Sentido , RNA Helicases/metabolismo , Transativadores/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Expansão das Repetições de DNA , Conjuntos de Dados como Assunto , Embrião de Mamíferos , Feminino , Lobo Frontal/patologia , Demência Frontotemporal/patologia , Humanos , Íntrons/genética , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Cultura Primária de Células , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA-Seq , Transativadores/genética
11.
Neurology ; 95(12): e1629-e1639, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32675077

RESUMO

OBJECTIVE: We implemented automated methods to analyze speech and evaluate the hypothesis that cognitive and motor factors impair prosody in partially distinct ways in patients with amyotrophic lateral sclerosis (ALS). METHODS: We recruited 213 participants, including 67 with ALS (44 with motor ALS, 23 with ALS and frontotemporal degeneration [FTD]), 33 healthy controls, and neurodegenerative reference groups with behavioral variant FTD (n = 90) and nonfluent/agrammatic primary progressive aphasia (n = 23). Digitized, semistructured speech samples obtained from picture descriptions were automatically segmented with a Speech Activity Detector; continuous speech segments were pitch-tracked; and duration measures for speech and silent pause segments were extracted. Acoustic measures were calculated, including fundamental frequency (f0) range, mean speech and pause segment durations, total speech duration, and pause rate (pause count per minute of speech). Group comparisons related performance on acoustic measures to clinical scales of cognitive and motor impairments and explored MRI cortical thinning in ALS and ALS-FTD. RESULTS: The f0 range was significantly impaired in ALS spectrum disorders and was related to bulbar motor disease, and regression analyses related this to cortical thickness in primary motor cortex and perisylvian regions. Impaired speech and pause duration measures were related to the degree of cognitive impairment in ALS spectrum disorders, and regressions related duration measures to bilateral frontal opercula and left anterior insula. CONCLUSION: Automated analyses of acoustic speech properties dissociate motor and cognitive components of speech deficits in ALS spectrum disorders.


Assuntos
Esclerose Amiotrófica Lateral/complicações , Encéfalo/diagnóstico por imagem , Distúrbios da Fala/diagnóstico por imagem , Medida da Produção da Fala/métodos , Idoso , Esclerose Amiotrófica Lateral/diagnóstico por imagem , Esclerose Amiotrófica Lateral/patologia , Encéfalo/patologia , Feminino , Humanos , Imagem por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Neuroimagem/métodos , Distúrbios da Fala/etiologia , Distúrbios da Fala/patologia
12.
Mol Cell ; 79(3): 443-458.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32649883

RESUMO

Despite the prominent role of TDP-43 in neurodegeneration, its physiological and pathological functions are not fully understood. Here, we report an unexpected role of TDP-43 in the formation of dynamic, reversible, liquid droplet-like nuclear bodies (NBs) in response to stress. Formation of NBs alleviates TDP-43-mediated cytotoxicity in mammalian cells and fly neurons. Super-resolution microscopy reveals distinct functions of the two RRMs in TDP-43 NB formation. TDP-43 NBs are partially colocalized with nuclear paraspeckles, whose scaffolding lncRNA NEAT1 is dramatically upregulated in stressed neurons. Moreover, increase of NEAT1 promotes TDP-43 liquid-liquid phase separation (LLPS) in vitro. Finally, we discover that the ALS-associated mutation D169G impairs the NEAT1-mediated TDP-43 LLPS and NB assembly, causing excessive cytoplasmic translocation of TDP-43 to form stress granules, which become phosphorylated TDP-43 cytoplasmic foci upon prolonged stress. Together, our findings suggest a stress-mitigating role and mechanism of TDP-43 NBs, whose dysfunction may be involved in ALS pathogenesis.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteínas de Ligação a DNA/genética , Corpos de Inclusão Intranuclear/metabolismo , Neurônios/metabolismo , RNA Longo não Codificante/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Animais Geneticamente Modificados , Arsenitos/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/ultraestrutura , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Corpos de Inclusão Intranuclear/efeitos dos fármacos , Corpos de Inclusão Intranuclear/ultraestrutura , Camundongos , Mutação , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Cultura Primária de Células , Transporte Proteico/efeitos dos fármacos , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Estresse Fisiológico
13.
Mol Cell ; 79(1): 54-67.e7, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32521226

RESUMO

Exposure of cells to heat or oxidative stress causes misfolding of proteins. To avoid toxic protein aggregation, cells have evolved nuclear and cytosolic protein quality control (PQC) systems. In response to proteotoxic stress, cells also limit protein synthesis by triggering transient storage of mRNAs and RNA-binding proteins (RBPs) in cytosolic stress granules (SGs). We demonstrate that the SUMO-targeted ubiquitin ligase (StUbL) pathway, which is part of the nuclear proteostasis network, regulates SG dynamics. We provide evidence that inactivation of SUMO deconjugases under proteotoxic stress initiates SUMO-primed, RNF4-dependent ubiquitylation of RBPs that typically condense into SGs. Impairment of SUMO-primed ubiquitylation drastically delays SG resolution upon stress release. Importantly, the StUbL system regulates compartmentalization of an amyotrophic lateral sclerosis (ALS)-associated FUS mutant in SGs. We propose that the StUbL system functions as surveillance pathway for aggregation-prone RBPs in the nucleus, thereby linking the nuclear and cytosolic axis of proteotoxic stress response.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Núcleo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína SUMO-1/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Esclerose Amiotrófica Lateral/genética , Núcleo Celular/genética , Células HeLa , Resposta ao Choque Térmico , Humanos , Mutação , Proteínas Nucleares/genética , Proteólise , Proteína FUS de Ligação a RNA/genética , Proteínas de Ligação a RNA/genética , Proteína SUMO-1/genética , Sumoilação , Fatores de Transcrição/genética , Ubiquitinação
14.
Proc Natl Acad Sci U S A ; 117(26): 15230-15241, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32513711

RESUMO

Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/genética , Autofagossomos/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Demência/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Proteínas Relacionadas à Autofagia/genética , Biomarcadores/metabolismo , Linhagem Celular , Demência/metabolismo , Demência/patologia , Predisposição Genética para Doença , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Ligação Proteica , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Regulação para Cima , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
16.
Nature ; 582(7810): 89-94, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32483373

RESUMO

A hexanucleotide-repeat expansion in C9ORF72 is the most common genetic variant that contributes to amyotrophic lateral sclerosis and frontotemporal dementia1,2. The C9ORF72 mutation acts through gain- and loss-of-function mechanisms to induce pathways that are implicated in neural degeneration3-9. The expansion is transcribed into a long repetitive RNA, which negatively sequesters RNA-binding proteins5 before its non-canonical translation into neural-toxic dipeptide proteins3,4. The failure of RNA polymerase to read through the mutation also reduces the abundance of the endogenous C9ORF72 gene product, which functions in endolysosomal pathways and suppresses systemic and neural inflammation6-9. Notably, the effects of the repeat expansion act with incomplete penetrance in families with a high prevalence of amyotrophic lateral sclerosis or frontotemporal dementia, indicating that either genetic or environmental factors modify the risk of disease for each individual. Identifying disease modifiers is of considerable translational interest, as it could suggest strategies to diminish the risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, or to slow progression. Here we report that an environment with reduced abundance of immune-stimulating bacteria10,11 protects C9orf72-mutant mice from premature mortality and significantly ameliorates their underlying systemic inflammation and autoimmunity. Consistent with C9orf72 functioning to prevent microbiota from inducing a pathological inflammatory response, we found that reducing the microbial burden in mutant mice with broad spectrum antibiotics-as well as transplanting gut microflora from a protective environment-attenuated inflammatory phenotypes, even after their onset. Our studies provide further evidence that the microbial composition of our gut has an important role in brain health and can interact in surprising ways with well-known genetic risk factors for disorders of the nervous system.


Assuntos
Proteína C9orf72/genética , Microbioma Gastrointestinal/fisiologia , Gliose/microbiologia , Gliose/patologia , Inflamação/genética , Inflamação/microbiologia , Medula Espinal/patologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Animais , Antibacterianos/farmacologia , Autoimunidade/efeitos dos fármacos , Autoimunidade/genética , Autoimunidade/imunologia , Movimento Celular/efeitos dos fármacos , Citocinas/imunologia , Transplante de Microbiota Fecal , Feminino , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/imunologia , Gliose/genética , Gliose/prevenção & controle , Inflamação/patologia , Inflamação/prevenção & controle , Mutação com Perda de Função/genética , Masculino , Camundongos , Microglia/imunologia , Microglia/microbiologia , Microglia/patologia , Medula Espinal/imunologia , Medula Espinal/microbiologia , Taxa de Sobrevida
17.
J Vis Exp ; (159)2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32478750

RESUMO

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.


Assuntos
Envelhecimento/patologia , Drosophila melanogaster/fisiologia , Degeneração Neural/patologia , Sinapses/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Animais Geneticamente Modificados , Denervação , Dissecação , Congelamento , Humanos , Larva/metabolismo , Junção Neuromuscular/fisiologia , Coloração e Rotulagem , Tórax
18.
Neurology ; 94(24): e2592-e2604, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32414878

RESUMO

OBJECTIVE: To understand the progressive nature of amyotrophic lateral sclerosis (ALS) by investigating differential brain patterns of gray and white matter involvement in clinically or genetically defined subgroups of patients using cross-sectional, longitudinal, and multimodal MRI. METHODS: We assessed cortical thickness, subcortical volumes, and white matter connectivity from T1-weighted and diffusion-weighted MRI in 292 patients with ALS (follow-up: n = 150) and 156 controls (follow-up: n = 72). Linear mixed-effects models were used to assess changes in structural brain measurements over time in patients compared to controls. RESULTS: Patients with a C9orf72 mutation (n = 24) showed widespread gray and white matter involvement at baseline, and extensive loss of white matter integrity in the connectome over time. In C9orf72-negative patients, we detected cortical thinning of motor and frontotemporal regions, and loss of white matter integrity of connections linked to the motor cortex. Patients with spinal onset displayed widespread white matter involvement at baseline and gray matter atrophy over time, whereas patients with bulbar onset started out with prominent gray matter involvement. Patients with unaffected cognition or behavior displayed predominantly motor system involvement, while widespread cerebral changes, including frontotemporal regions with progressive white matter involvement over time, were associated with impaired behavior or cognition. Progressive loss of gray and white matter integrity typically occurred in patients with shorter disease durations (<13 months), independent of progression rate. CONCLUSIONS: Heterogeneity of phenotype and C9orf72 genotype relates to distinct patterns of cerebral degeneration. We demonstrate that imaging studies have the potential to monitor disease progression and early intervention may be required to limit cerebral degeneration.


Assuntos
Esclerose Amiotrófica Lateral/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Idoso , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Comportamento , Encéfalo/patologia , Proteína C9orf72/genética , Cognição , Estudos Transversais , Imagem de Difusão por Ressonância Magnética , Progressão da Doença , Feminino , Substância Cinzenta/diagnóstico por imagem , Substância Cinzenta/patologia , Humanos , Estudos Longitudinais , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Imagem Multimodal , Mutação , Estudos Prospectivos , Substância Branca/diagnóstico por imagem , Substância Branca/patologia
19.
Adv Exp Med Biol ; 1241: 195-217, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32383122

RESUMO

Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative diseases, characterized by inevitable progressive paralysis. To date, only two disease modifying therapeutic options are available for the patients with ALS, although they show very modest effect on disease course. The main reason of failure in the field of pharmacological correction of ALS is inability to untangle complex relationships taking place during ALS initiation and progression. Traditional methods of research, based on morphology or transgenic animal models studying provided lots of information about ALS throughout the years. However, translation of these results to humans was unsuccessful due to incomplete recapitulation of molecular pathology and overall inadequacy of the models used in the research.In this review we summarize current knowledge regarding ALS molecular pathology with depiction of novel methods applied recently for the studies. Furthermore we describe present and potential treatment strategies that are based on the recent findings in ALS disease mechanisms.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Pesquisa Biomédica , Progressão da Doença , Humanos
20.
Neuron ; 107(1): 95-111.e6, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32380032

RESUMO

Progressive synapse loss is an inevitable and insidious part of age-related neurodegenerative disease. Typically, synapse loss precedes symptoms of cognitive and motor decline. This suggests the existence of compensatory mechanisms that can temporarily counteract the effects of ongoing neurodegeneration. Here, we demonstrate that presynaptic homeostatic plasticity (PHP) is induced at degenerating neuromuscular junctions, mediated by an evolutionarily conserved activity of presynaptic ENaC channels in both Drosophila and mouse. To assess the consequence of eliminating PHP in a mouse model of ALS-like degeneration, we generated a motoneuron-specific deletion of Scnn1a, encoding the ENaC channel alpha subunit. We show that Scnn1a is essential for PHP without adversely affecting baseline neural function or lifespan. However, Scnn1a knockout in a degeneration-causing mutant background accelerated motoneuron loss and disease progression to twice the rate observed in littermate controls with intact PHP. We propose a model of neuroprotective homeostatic plasticity, extending organismal lifespan and health span.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Homeostase/fisiologia , Plasticidade Neuronal/fisiologia , Neuroproteção/fisiologia , Terminações Pré-Sinápticas/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Drosophila melanogaster , Camundongos , Camundongos Knockout , Junção Neuromuscular/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA